Platelets, which play a role in hemostasis, are of three general types: megakaryocytes, reticulated (i.e., immature) platelets that are released into the peripheral blood following megakaryocytic fragmentation, and mature platelets. However, other subpopulations of platelets exist in the blood, which are commonly uncounted or miscounted in typical clinical analyses. Such ‘subpopulations’ include giant platelets (i.e., unusually large platelets), platelet clumps (i.e., multi-platelet combinations), and platelet satellites (i.e., adherence of platelets to leucocytes or other cells).
The differentiation and enumeration of these various types of blood cells and platelets in a patient's peripheral blood, the ratios thereof, as well as the determination of certain parameters or characteristics thereof, are necessary to permit diagnosis of a variety of hematological disorders or diseases. The absolute numbers, concentrations and relative percentages of the different types of platelets are highly indicative of the presence or absence and/or stage of certain disease states. For example, the measurement and enumeration of platelet types is important for the diagnosis and monitoring of a variety of disorders characterized by abnormal presence and/or numbers of platelets at various stages of maturation. For example, the identification and measurement of the platelet populations individually, the ratio thereof, as well as an accurate measurement of total platelet numbers including all subpopulations, has considerable clinical utility for monitoring thrombopoiesis and platelet turnover.
Typically, only mature cells are present in detectable amounts in peripheral blood. When a platelet count is suspiciously low, and/or the automated blood cell counter presents with a warning, a manual blood smear is typically the only way to determine whether or not there are adequate platelets present and to verify the presence of giant platelets, and/or platelet clumps. Manual counting involves contacting the cells with nucleic acid specific, non-metachromatic dyes (Kienast, J. and Schmitz, G. 1990 Blood 75: 116-121; Ault, K. A. et al, 1992 Am J Clin Pathol 98: 637-646; Robinson, M. S. C. et al, 1998 Brit J Haematol 100: 351-357) or nucleic acid specific, metachromatic dyes (Schmitz, F. J. and Werner, E., 1986 Cytometry 7: 439-444; and U.S. Pat. No. 6,060,322). Metachromatic dyes emit fluorescence over a broad range of wavelengths and are particularly useful for staining immature reticulated platelets, which contain RNA condensed as reticulum. For example, on histochemical staining with new methylene blue, the reticulum of immature platelets appears as patchy purplish-blue areas. Once stained, all the platelets are enumerated in a microscopic view, expressing the platelets containing the reticulum as a percentage of the total platelets and expressing them as a percentage of reticulated platelets (Ingram, M. and Coopersmith, A. 1969 Brit J Haematol 17: 225-228). This manual counting procedure is tedious, cumbersome and prone to human counting errors.
Another method of platelet analysis employs flow cytometry techniques, which are faster and more reliable. Such techniques generally employ the non-metachromatic dyes, thiazole orange (TO), auramine O, and polymethine with oxazine (Lee, L. G. et al, 1986 Cytometry 7: 508-517; Watanabe, K., et al, 1995 Eur J Haematol 54: 163-171; Briggs, C. et al, 2004 Brit L Haematol 126: 93-99). Acridine orange (AO), a metachromatic dye, is also used (Seligman, P. A. et al, 1983 Am. J Hematol 14: 57-66; U.S. Pat. No. 6,060,322). These methods, although useful for measuring mature platelets and reticulated platelets, suffer from certain drawbacks. These methods use size (log forward scatter) and granularity (log side scatter) to identify and resolve the platelets from the rest of the blood components. The broad nature of the metachromatic dye's fluorescence emission precluded the use of fluorochrome conjugated antibodies to identify the platelets.
In blood samples from healthy volunteers, the use of size and granularity is sufficient to include only platelets in the measurement. However, samples from patients with certain diseases have contaminating red cell fragments that fall within the platelet population, causing the platelet area to have an apparent increase in the number of platelets therefore leading to a lower percentage of reticulated platelets. Additionally, where giant platelets, platelet clumps or platelet satellites are present due to a platelet related deficiency or abnormality, the resulting identification or population or quantification can be incorrectly determined by the flow cytometer.
Another compounding issue in conventional flow cytometry measurement methods using nucleic acid dyes is the low platelet count in blood samples obtained from some patients. One example is the disorder called idiopathic thrombocytopenic purpura, in which the patients' own platelets are coated with an autoantibody and removed by the mononuclear phagocyte system. In order to count enough platelets for a statistically valid reticulated platelet percentage in this disease, one must acquire a huge data file with the bulk of the counts still being that of red blood cells.
Such automated flow cytometric methods have also employed platelet-specific antibodies in conjunction with non-metachromatic nucleic acid dyes for the purpose of measuring reticulated platelets. See, for example, Bonan et al 1993 Cytometry, 14:690-694; Matic et al 1998 Cytometry, 34:229-234; Rinder et al 1998, Blood, 91:1288-1294; Robinson et al, 1998, cited above; Balduini, C. L., et al, 1999 Brit J Haematol 106: 202-207; Stohlawetz, P., et al 1999 Thromb Haemost 81: 613-617; Saxonhouse, M. A. et al, 2004 J Pediatr Hematol Oncol: 26, 797-802; Chaoui, D. et al., 2005 Transfusion: 45, 766-772). As an example, fluorochrome conjugated antibodies with peak emissions beyond 560 nm are used in conjunction with the dye thiazole orange (TO), which binds to RNA on excitation with a 488 nm laser and has detectable emission from 500 to 560 nm, by using appropriate optical filter and applying fluorescence compensation (Saxonhouse et al 2004, cited above). However, other dyes do not work in this method. For example, the metachromatic dye AO, when bound to RNA, has a broad overlapping emission from 500 nm to 700 nm with the peak around 630 to 670 nm. It is therefore difficult to use AO with fluorochrome conjugated antibodies with emission spectra that span that range, even if fluorescence compensation is also used (Shapiro, H. M. 2003 Nucleic Acid Dyes and their Uses. In Practical Flow Cytometry, 4th Edition, H M Shapiro (Ed), John Wiley & Sons, Hoboken, N.J., pp 306-326).
In general, such flow cytometric methods involve lengthy incubation times; and thus such methods are not amenable for use on high throughput hematology systems. This is exemplified in the measurement of reticulated platelets in whole blood with TO and a platelet specific antibody. An anti-CD41 platelet specific antibody was reported in one publication using an incubation time of 15 minutes (Chaoui et al 2005, cited above). The same reagents were used in other methods, but with different incubation times of 30 and 45 minutes respectively (Stohlawetz et al 1999 and Saxonhouse et al, 2004, both cited above). Additional publications using TO and fluorochrome conjugated anti-platelet antibody use incubation times for the antibodies to bind to platelets ranging from 10 to 45 minutes (Bonan et al 1993, Matic et a 1998, Rinder et al 1998, Robinson et al, 1998, Balduini et al 1999, all cited above). These tong incubation times render these assays useless for high throughput hematology systems.
Similarly, in the wider area of nucleic acid dye binding and surface staining with antibodies for blood components other than platelets, the incubation times are similarly long, e.g., 30 minutes (U.S. Pat. No. 5,047,321); and 15 minutes (U.S. Pat. No. 6,287,791) and thus not suitable for automated cell analysis.
There remains a need in the art for rapid analytic methods that enhance the specific identification of platelets and reticulated platelets in clinical situations where low platelet counts or interfering conditions can lead to inaccuracies of the measurement. There also remains a need for such methods that are suitable for performance in an automated hematology analyzer without compromising the throughput of the instrument.